In 1966, Ishizaka established that the human allergen reaginic antibodies belong to a distinct class of immunoglobulins, IgE. There followed a decade of remarkable sequence of events. Sensitization of the human and monkey skin to Prausnitz-Kunster reaction by IgE antibodies was demonstrated. It was recognized that the role of IgE is central to the release mechanisms of chemical mediators of anaphylaxis; it mediates immunologic release of histamine from human leucocytes and mast cells; it primes the human lung tissue for antigen-induced release of histamine and Slow Reacting Substance of Anaphylaxis; it triggers the release of eosinophil chemotactic factor from human lung; and its avidity for attachment, through its Fc portion, to the receptors on the surface of mast cells and basophil granules was shown. It has been inferred that the number and the affinity of IgE antibodies bound to the basophil granulocytes determines the sensitivity of this cell to the allergen, while the histamine release induced by the antigen-antibody reaction on the cell surface is the function of the intracellular enzyme system and cyclic AMP level.
Dating back to the early part of the 20th century, patients with hay fever were treated with injections of incriminated allergens, albeit without understanding the pathologic bases of the disease or the pharmacologic bases for the efficacy of the therapy. Johansson's observation, in 1967, of augmented levels of serum IgE in atopic patients sparked intense interest in this relationship; individuals with inhalant allergies were found to display seasonal peaks in their serum IgE levels; abatement of allergic symptomatology with immunotherapy was documented. Partial suppression of seasonal peaks following specific immunotherapy was demonstrated and the inter-relationship of levels of IgE and IgG in atopic subjects, and the changes induced by specific immunotherapy have been illuminated.
The major in vitro test used to determine IgE today is a radioimmunoassay technique known as the Radio Allergo Sorbant Test or RAST.
The advent of enzyme-labelled antibodies has been a major event in the progress of immunoassays. The use of such enzyme labels, for this purpose introduced in 1971, offers several advantages over radioimmunoassay techniques including the freedom from hazards of radioactive material, the stability of a label for months and possibly longer, the use of photometric rather than radiometric equipment, and at times, elimination of separation procedures. For these reasons, enzyme immunoassays have found wide-spread and diversified application both in reasearch and in clinical practice.
In 1966, Nakane & Pierce published a report demonstrating that peroxidase could be coupled to an antibody by a simple procedure to produce a stable conjugate. The intact immunological reactivity of such a conjugate was shown to render it eminently suitable for use in immunotracing methods, in a fashion similar to that of fluorescein-labelled antibody. Since that time a number of conjugates have been developed and used for enzymatic immunological tests. See, e.g., U.S. Pat. Nos. 4,016,043 and 3,645,852.
For the assay of total IgE in serum, alkaline phosphatase has been employed as the enzyme marker in an application of the enzyme linked immunosorbant assay and in a magnetic enzyme imminoassay. The use of both alkaline phosphatase and galactosidase has been described for the assay of allergen-specific IgE antibodies.
The success in terms of quantitation, sensitivity and absence of non-specific reaction in any solid phase immunoenzymatic technique depends, to a great extent, on the quality of the enzyme-antibody conjugate. The immunoenzymatic techniques described in the literature for use with IgE have been performed using conjugates prepared by means of bifunctional reagents. Thus, the use of alkaline phosphatase (Mol. Wt. 60,000) or galactosidase (molecular weight 580,000) as enzyme markers has required the use of glutaraldhyde for conjugation of the enzyme to the antibody. The negative controls of such procedures exhibit a high degree of background activity or interference which interferes with the readability, i.e., interpretation, of the test results. While no precise quantitation has been reported, we have found that the use of alkaline phosphate and galactosidase as enzyme markers has resulted in the final yield of a very small amount of functionally usable conjugates, usually 30% or less, and unacceptable contamination with large amounts of side reaction products. Such byproducts include enzyme-enzyme conjugates, IgG-IgG conjugates and large aggregates. The separation of the usable conjugate from the undesirable side reaction products has been tedious and time consuming and, in addition, the stability of the purified fraction has not been satisfactory.
It has now been determined that an in vitro solid phase immunoenzymatic allergy test for the presence of IgE which is very successful in terms of quantification, sensitivity and absence of non-specific reaction can be realized if the enzyme employed is Horse radish peroxidase (HRPO, molecular weight 40,000) and if the conjugate is prepared using enzyme with its free amino groups blocked with phenylisothiocyanate as the first step, and if the reduction of the conjugate is carried out by titration. When the test is carried out according to the method of the present invention, the results are such that a safe initial hypersensitization dose can be determined from the results.
Accordingly, it is the object of this invention to provide a new and improved method and reagents for carrying out an immunoenzymatic test for the presence of allergen specific IgE and for the use of the test results in hypersensitization therapy. This and other objects of the invention will become apparent to those skilled in this art from the following detailed description.